RESUMEN
The toxic nature of bacterial endotoxins is affected by the structural details of lipid A, including the variety and position of acyl chains and phosphate group(s) on its diglucosamine backbone. Negative-ion mode tandem mass spectrometry is a primary method for the structure elucidation of lipid A, used independently or in combination with separation techniques. However, it is challenging to accurately characterize constitutional isomers of lipid A extracts by direct mass spectrometry, as the elemental composition and molecular mass of these molecules are identical. Thus, their simultaneous fragmentation leads to a composite, so-called chimera mass spectrum. The present study focuses on the phosphopositional isomers of the classical monophosphorylated, hexaacylated Escherichia coli-type lipid A. Collision-induced dissociation (CID) was performed in an HPLC-ESI-QTOF system. Energy-resolved mass spectrometry (ERMS) was applied to uncover the distinct fragmentation profiles of the phosphorylation isomers. A fragmentation strategy applying multi-levels of collision energy has been proposed and applied to reveal sample complexity, whether it contains only a 4'-phosphorylated species or a mixture of 1- and 4'-phosphorylated variants. This comparative fragmentation study of isomeric lipid A species demonstrates the high potential of ERMS-derived information for the successful discrimination of co-ionized phosphorylation isomers of hexaacylated lipid A.
Asunto(s)
Lípido A , Espectrometría de Masas en Tándem , Lípido A/química , Isomerismo , Espectrometría de Masa por Ionización de Electrospray , Escherichia coli , Cromatografía Líquida de Alta Presión , FosforilaciónRESUMEN
Lipid A, the inflammatory portion of lipopolysaccharides (LPS, endotoxins), is the main component of the outer membrane of Gram-negative bacteria. Its bioactivity in humans and animals is strictly related to its chemical structure. In the present work, the fragmentation patterns of the singly charged monosodium [M + Na]+ and disodium [M - H + 2Na]+ adducts, as well as the protonated form of monophosphorylated lipid A species were investigated in detail using positive-ion electrospray ionization-based tandem (MS/MS) and multistage mass spectrometry (MSn) with low-energy collision-induced dissociation (CID). Several synthetic and native lipid A samples were included in the study. We found that the fragmentation pattern of disodiated lipid A is quite similar to that of the well-characterized deprotonated lipid A molecule (typically detected in the negative-ion mode), while the fragmentation pattern of monosodiated lipid A contains fragment ions similar to those of both protonated and deprotonated lipid A molecules. In summary, we propose a new mass spectrometry approach based on the fragmentation regularities of only positively charged precursor ions to dissect the location of the phosphate group and fatty acid moieties on monophosphorylated lipid A. Moreover, this study provides a better understanding of the so-called "chimera mass spectra", which are commonly detected during the fragmentation of native lipid A samples containing both C-1 and C-4' phosphate positional isomers but rarely identified in negative-ion mode.
Asunto(s)
Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masas en Tándem , Humanos , Espectrometría de Masas en Tándem/métodos , Espectrometría de Masa por Ionización de Electrospray/métodos , Lípido A , Isomerismo , Lipopolisacáridos , IonesRESUMEN
This paper presents the genome sequence of a Shigella sonnei mutant strain (S. sonnei 4351) and the effect of mutation in lipopolysaccharide biosynthesis on bacterial fitness. Lipopolysaccharides are the major component of the outer leaflet of the Gram-negative outer membrane. We report here a frameshift mutation of the gene gmhD in the genome of S. sonnei 4351. The mutation results in a lack of epimerization of the core heptose while we also found increased thermosensitivity, abnormal cell division, and increased susceptibility to erythromycin and cefalexin compared to the S. sonnei 4303. Comparative genomic analysis supplemented with structural data helps us to understand the effect of specific mutations on the virulence of the bacteria and may provide an opportunity to study the effect of short lipopolysaccharides.
Asunto(s)
Aptitud Genética , Lipopolisacáridos , Shigella sonnei , Cefalexina/farmacología , Eritromicina/farmacología , Lipopolisacáridos/genética , Shigella sonnei/efectos de los fármacos , Shigella sonnei/genética , Genoma Bacteriano , Antibacterianos/farmacología , Carbohidrato Epimerasas/genética , Proteínas Bacterianas/genética , Mutación del Sistema de LecturaRESUMEN
Lipid A, the membrane-bound phosphoglycolipid component of bacteria, is held responsible for the clinical syndrome of gram-negative sepsis. In this study, the fragmentation behavior of a set of synthetic lipid A derivatives was studied by electrospray ionization multistage mass spectrometry (ESI-MSn), in conjunction with tandem mass spectrometry (MS/MS), using low-energy collision-induced dissociation (CID). Genealogical insight about the fragmentation pathways of the deprotonated 4'-monophosphoryl lipid A structural analogs led to proposals of a number of alternative dissociation routes that have not been reported previously. Each of the fragment ions was interpreted using various possible mechanisms, consistent with the principles of reactions described in organic chemistry. Specifically, the hypothesized mechanisms are: (i) cleavage of the C-3 primary fatty acid leaves behind an epoxide group attached to the reducing sugar; (ii) cleavage of the C-3' primary fatty acid (as an acid) generates a cyclic phosphate connected to the nonreducing sugar; (iii) cleavage of the C-2' secondary fatty acid occurs both in acid and ketene forms; iv) the C-2 and C-2' primary fatty acids are eliminated as an amide and ketene, respectively; (v) the 0,2A2 cross-ring fragment contains a four-membered ring (oxetanose); (vi) the 0,4A2 ion is consecutively formed from the 0,2A2 ion by retro-aldol, retro-cycloaddition, and transesterification; and (vii) formations of H2PO4- and PO3- are associated with the formation of sugar epoxide. An understanding of the relation between 0,2A2 and 0,4A2-type sugar fragments and the different cleavage mechanisms of the two ester-linked primary fatty acids is invaluable for distinguishing lipid A isomers with different locations of a single ester-linked fatty acid (i.e., at C-3 or C-3'). Thus, in addition to a better comprehension of lipid A fragmentation processes in mass spectrometers, our observations can be applied for a more precise elucidation of naturally occurring lipid A structures.
Asunto(s)
Ácidos Grasos/química , Lípido A/análogos & derivados , Reacción de Cicloadición , Esterificación , Lípido A/química , Fosforilación , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masas en TándemRESUMEN
Lipid A represents a heterogeneous group of bacterial outer membrane phosphoglycolipids, which play a major role in the pathogenesis of Gram-negative sepsis. The number and position of phosphoryl and acyl groups in lipid A molecules are key structural determinants in their bioactivities. In this study, a NACE-ESI-MS/MS method was developed for the simultaneous analysis of lipid A isomers possessing a different degree of phosphorylation and acylation. Various C4'- and C1-monophosphorylated lipid A isobars, as well as acylation isomers, were baseline separated within 43 min in a separation medium of methanol/dichloromethane/triethylamine/acetic acid 60:40:1.08:0.36 (v/v/v/v). Both normal and reverse CE polarities could be applied for proper detection of the analytes owing to the combination of a suction effect caused by the nebulizer gas at the outlet end of the capillary and external pressure applied on the inlet vial. The separated lipid A species could be identified unequivocally by their characteristic fragmentation patterns through CID performed in both negative- and positive-ionization modes. The uniqueness of the NACE-ESI-MS/MS method lies in its simplicity and reliability for proving the phosphorylation isomerism (C1 or C4') and acylation pattern of native lipid A species or those designed for therapeutic applications.
Asunto(s)
Electroforesis Capilar/métodos , Lípido A/química , Lípido A/aislamiento & purificación , Espectrometría de Masa por Ionización de Electrospray/métodos , Espectrometría de Masas en Tándem/métodos , Acilación , Isomerismo , Fosforilación , Shigella sonnei/químicaRESUMEN
OBJECTIVES: Regulation of lipopolysaccharide (LPS) chemical composition, particularly its lipid A domain, is an important, naturally occurring mechanism that drives bacteria-host immune system interactions into either a symbiotic or pathogenic relationship. Members of the subgingival oral microbiota can critically modulate host immuno-inflammatory responses by synthesizing different LPS isoforms. The objectives of this study were to analyze subgingival lipid A profiles and endotoxin activities in periodontal health and disease and to evaluate the use of the recombinant factor C assay as a new, lipid A-based biosensor for personalized, point-of-care periodontal therapy. MATERIALS AND METHODS: Subgingival plaque samples were collected from healthy individuals and chronic periodontitis patients before and after periodontal therapy. Chemical composition of subgingival lipid A moieties was determined by ESI-Mass Spectrometry. Endotoxin activity of subgingival LPS extracts was assessed using the recombinant factor C assay, and their inflammatory potential was examined in THP-1-derived macrophages by measuring TNF-α and IL-8 production. RESULTS: Characteristic lipid A molecular signatures, corresponding to over-acylated, bi-phosphorylated lipid A isoforms, were observed in diseased samples. Healthy and post-treatment samples were characterized by lower m/z peaks, related to under-acylated, hypo-phosphorylated lipid A structures. Endotoxin activity levels and inflammatory potentials of subgingival LPS extracts from periodontitis patients were significantly higher compared to healthy and post-treatment samples. CONCLUSIONS: This is the first study to consider structure-function-clinical implications of different lipid A isoforms present in the subgingival niche and sheds new light on molecular pathogenic mechanisms of subgingival biofilm communities. CLINICAL RELEVANCE: Subgingival endotoxin activity (determined by lipid A chemical composition) could be a reliable, bacterially derived biomarker and a risk assessment tool for personalized periodontal care.
Asunto(s)
Periodontitis Crónica , Placa Dental , Endotoxinas , Microbiota , Periodontitis , Bacterias , Placa Dental/metabolismo , Placa Dental/microbiología , Endotoxinas/metabolismo , Humanos , Lípido A/metabolismo , Periodontitis/metabolismo , Periodontitis/microbiologíaRESUMEN
In this study, we report the detailed analysis of the fragmentation patterns of positively charged lipid A species based on their tandem mass spectra obtained under low-energy collision-induced dissociation conditions of an electrospray quadrupole time-of-flight mass spectrometer. The tandem mass spectrometry experiments were performed after the separation of the compounds with a reversed-phase high performance liquid chromatography method. We found that both, phosphorylated and nonphosphorylated lipid A molecules can be readily ionized in the positive-ion mode by adduct formation with triethylamine added to the eluent. The tandem mass spectra of the lipid A triethylammonium adduct ions showed several product ions corresponding to inter-ring glycosidic cleavages of the sugar residues, as well as consecutive and competitive eliminations of fatty acids, phosphoric acid, and water following the neutral loss of triethylamine. Characteristic product ions provided direct information on the phosphorylation site(s), also when phosphorylation isomers (ie, containing either a C1 or a C4' phosphate group) were simultaneously present in the sample. Continuous series of high-abundance B-type and low-abundance Y-type inter-ring fragment ions were indicative of the fatty acyl distribution between the nonreducing and reducing ends of the lipid A backbone. The previously reported lipid A structures of Proteus morganii O34 and Escherichia coli O111 bacteria were used as standards. Although, the fragmentation pathways of the differently phosphorylated lipid A species significantly differed in the negative-ion mode, they were very similar in the positive-ion mode. The complementary use of positive-ion and negative-ion mode tandem mass spectrometry was found to be essential for the full structural characterization of the C1-monophosphorylated lipid A species.
Asunto(s)
Lípido A/química , Acilación , Cromatografía Líquida de Alta Presión/métodos , Escherichia coli/química , Estructura Molecular , Fosforilación , Polisacáridos/química , Proteus/química , Espectrometría de Masa por Ionización de Electrospray/métodos , Espectrometría de Masas en TándemRESUMEN
Endotoxins (lipopolysaccharides, LPS; lipooligosaccharides, LOS) are components of the envelope of Gram-negative bacteria. These molecules, responsible for both advantageous and harmful biological activity of these microorganisms, are highly immunogenic and directly involved in numerous bacterial diseases in humans, such as Gram-negative sepsis. The characterization of endotoxins is of importance, since their physiological and pathophysiological effects depend on their chemical structure. The differences among the LPS from different bacterial serotypes and their mutants include variations mainly within the composition and length or missing of their O-polysaccharide chains. Microchip electrophoretic methodology enables the structural characterization of LPS molecules from several bacteria and the quantitative evaluation of components of endotoxin extracts. The improved microchip electrophoretic method is based on the direct labeling of endotoxins by covalent binding of a fluorescent dye. The classification of the S-type LPSs can be done according to their electrophoretic profiles, which are characteristics of the respective bacterial strains. According to the number, distribution, and the relative amounts of components in an endotoxin extract, it is possible to differentiate between the S-type endotoxins from different Gram-negative bacterial strains. The microchip electrophoresis affords high-resolution separation of pure and partially purified (e.g., obtained from whole-cell lysate) S and R endotoxins. This microchip technique provides a new, standardizable, fast, and sensitive method for the detection of endotoxins and for the quantitative evaluation of components of an endotoxin extract.
Asunto(s)
Electroforesis Capilar/métodos , Endotoxinas/análisis , Lipopolisacáridos/análisisRESUMEN
Lipopolysaccharides (LPSs, endotoxins) are components of the outer cell membrane of most Gram-negative bacteria and can play an important role in a number of diseases of bacteria, including Gram-negative sepsis. The hydrophilic carbohydrate part of LPSs consists of a core oligosaccharide (in the case of an R-type LPS or lipooligosaccharide, LOS) linked to an O-polysaccharide chain (in the case of an S-type LPS), which is responsible for O-specific immunogenicity. The hydrophobic lipid A anchor is composed of a phosphorylated diglucosamine backbone to which varying numbers of ester- and amide-linked fatty acids are attached and this part of the LPSs is associated with endotoxicity. The detailed chemical characterization of endotoxins requires long-lasting large-scale isolation procedures, by which high-purity LPSs can be obtained. However, when a large number of bacterial samples and their LPS content are to be compared prompt, small-scale isolation methods are used for the preparation of endotoxins directly from bacterial cell cultures. The purity of the endotoxins extracted by these methods may not be high, but it is sufficient for analysis.Here, we describe a fast and easy micromethod suitable for extracting small quantities of LOS and a slightly modified micromethod for the detection of the lipid A constituents of the LPSs from bacteria grown in different culture media and evaluate the structures with mass spectrometry. The cellular LOS and lipid A were obtained from crude isolates of heat-killed cells, which were then subjected to matrix-assisted laser desorption/ionization mass spectrometry analysis. The observed ions in the 10-colony samples were similar to those detected for purified samples. The total time for the sample preparation and the MS analysis is less than 3 h.
Asunto(s)
Lípido A/química , Lipopolisacáridos/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Oligosacáridos/químicaRESUMEN
We established a new reversed phase-high performance liquid chromatography method combined with electrospray ionization quadrupole time-of-flight tandem mass spectrometry for the simultaneous determination and structural characterization of different lipid A types in bacteria (Escherichia coli O111, Salmonella adelaide O35 and Proteus morganii O34) showing serological cross-reactivity. The complex lipid A mixtures (obtained by simple extraction and acid hydrolysis of the outer membrane lipopolysaccharides) were separated and detected without phosphate derivatization. Several previously unidentified ions were detected, which differed in the number and type of acyl chains and number of phosphate groups. In several cases, we observed the different retention of isobaric lipid A species, which had different secondary fatty acyl distribution at the C2' or the C3' sites. The fragmentation of the various, C4' monophosphorylated lipid A species in deprotonated forms provided structural assignment for each component. Fragmentation pathways of the tri-acylated, tetra-acylated, penta-acylated, hexa-acylated and hepta-acylated lipid A components and of the lipid A partial structures are suggested. As standards, the hexa-acylated ion at m/z 1716 with the E. coli-type acyl distribution and the hepta-acylated ion at m/z 1954 with the Salmonella-type acyl distribution were used. The results confirmed the presence of multiple forms of lipid A in all strains analyzed. In addition, the negative-ion mode MS permitted efficient detection for non-phosphorylated lipid A components, too. Copyright © 2016 John Wiley & Sons, Ltd.
Asunto(s)
Lípido A/química , Acilación , Cromatografía Líquida de Alta Presión/métodos , Escherichia coli/química , Hidrólisis , Fosforilación , Proteolisis , Proteus/química , Salmonella/química , Espectrometría de Masa por Ionización de Electrospray/métodos , Espectrometría de Masas en Tándem/métodosRESUMEN
Non-phosphorylated lipid A species confer reduced inflammatory potential for the bacteria. Knowledge on their chemical structure and presence in bacterial pathogens may contribute to the understanding of bacterial resistance and activation of the host innate immune system. In this study, we report the fragmentation pathways of negatively charged, non-phosphorylated lipid A species under low-energy collision-induced dissociation conditions of an electrospray ionization quadrupole time-of-flight instrument. Charge-promoted consecutive and competitive eliminations of the acyl chains and cross-ring cleavages of the sugar residues were observed. The A-type fragment ion series and the complementary X-type fragment(s) with corresponding deprotonated carboxamide(s) were diagnostic for the distribution of the primary and secondary acyl residues on the non-reducing and the reducing ends, respectively, of the non-phosphorylated lipid A backbone. Reversed-phase liquid chromatography in combination with negative-ion electrospray ionization quadrupole time-of-flight tandem mass spectrometry could provide sufficient information on the primary and secondary acyl residues of a non-phosphorylated lipid A. As a standard, the hexa-acylated ion at m/z 1636 with the Escherichia coli-type acyl distribution (from E. coli O111) was used. The method was tested and refined with the analysis of other non-phosphorylated hexa- and several hepta-, penta-, and tetra-acylated lipid A species detected in crude lipid A fractions from E. coli O111 and Proteus morganii O34 bacteria. Copyright © 2016 John Wiley & Sons, Ltd.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Lípido A/análisis , Lípido A/química , Espectrometría de Masas en Tándem/métodos , Escherichia coli/química , Modelos Moleculares , Fosforilación , Proteus/químicaRESUMEN
The structure of the oligosaccharide repeating units of endotoxins from Gram-negative bacteria is characteristic for the different serogroups and serotypes of bacteria. Detailed examination of the cross-reactions of three enterobacterial serotypes, Proteus morganii O34, Escherichia coli O111, and Salmonella enterica sv. Adelaide O35, was performed using sensitive tests (ELISA, immunoblotting). Fine differences between the endotoxins of the bacteria were detected using silver staining of SDS-PAGE gels and chip-technology for the intact lipopolysaccharides (LPSs). The compositions of the O-specific polysaccharides of LPSs extracted from the bacteria were studied, and it was proven that the three cross-reacting bacteria contain O-antigens built from the same monosaccharides, namely colitoses linked to glucose, galactose, and N-acetyl-galactosamine. The NMR and GC-MS studies revealed that the most probable component for the cross-reaction is the rare sugar, colitose.
Asunto(s)
Reacciones Cruzadas , Enterobacteriaceae/inmunología , Conformación de Carbohidratos , Electroforesis en Gel de Poliacrilamida , Cromatografía de Gases y Espectrometría de Masas , Lipopolisacáridos/química , Resonancia Magnética Nuclear BiomolecularRESUMEN
With the need for high-frequency data acquisition, the influence of the data acquisition rate on the quality of the digitized signal is often discussed and also misinterpreted. In this study we show that undersampling of the signal, i.e. low data acquisition rate will not cause band broadening. Users of modern instrumentation and authors are frequently misled by hidden features of the data handling software they use. Very often users are unaware of the noise filtering algorithms that run parallel with data acquisition and that lack of information misleads them. We also demonstrate that undersampled signals can be restored by a proper trigonometric interpolation.
RESUMEN
The focus of this review is the application of mass spectrometry to the structural characterization of bacterial lipopolysaccharides (LPSs), also referred to as "endotoxins," because they elicit the strong immune response in infected organisms. Recently, a wide variety of MS-based applications have been implemented to the structure elucidation of LPS. Methodological improvements, as well as on- and off-line separation procedures, proved the versatility of mass spectrometry to study complex LPS mixtures. Special attention is given in the review to the tandem mass spectrometric methods and protocols for the analyses of lipid A, the endotoxic principle of LPS. We compare and evaluate the different ionization techniques (MALDI, ESI) in view of their use in intact R- and S-type LPS and lipid A studies. Methods for sample preparation of LPS prior to mass spectrometric analysis are also described. The direct identification of intrinsic heterogeneities of most intact LPS and lipid A preparations is a particular challenge, for which separation techniques (e.g., TLC, slab-PAGE, CE, GC, HPLC) combined with mass spectrometry are often necessary. A brief summary of these combined methodologies to profile LPS molecular species is provided.
Asunto(s)
Técnicas de Química Analítica , Lipopolisacáridos/química , Acilación , Cromatografía Líquida de Alta Presión , Cromatografía en Capa Delgada , Electroforesis Capilar , Electroforesis en Gel de Poliacrilamida , Cromatografía de Gases y Espectrometría de Masas , Isomerismo , Lipopolisacáridos/aislamiento & purificación , Estructura Molecular , Fosforilación , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Espectrometría de Masas en TándemRESUMEN
A novel, fast, and sensitive ME method was developed to analyze and differentiate the smooth (S) and rough (R) type bacterial endotoxin components labeled covalently with a fluorescent dye. The quantitative analysis of purified lipopolysaccharides, or partially purified samples from whole-cell lysates becomes possible with this method. Two groups with three sub-groups in the first group of S-type lipopolysaccharides can be classified based on the electrophoretic profiles. The LOD of the endotoxins from S- and R-type Gram-negative bacteria was found to be 2.6 ng and 6.9 ng, respectively. This method is capable to replace the commonly used SDS-PAGE combined with silver staining.
Asunto(s)
Electroforesis por Microchip/métodos , Lipopolisacáridos/análisis , Enterobacteriaceae/química , Colorantes Fluorescentes/química , Límite de Detección , Lipopolisacáridos/química , Lipopolisacáridos/aislamiento & purificación , Peso Molecular , Reproducibilidad de los Resultados , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , EstereoisomerismoRESUMEN
Endotoxins (lipopolysaccharides, LPSs) are components of the envelope of Gram-negative bacteria. These molecules, responsible for both advantageous and harmful biological activities of these microorganisms, are highly immunogenic and directly involved in numerous bacterial diseases in humans such as Gram-negative sepsis. The characterization of endotoxins is of importance, since their physiological and pathophysiological effects depend on their chemical structure. The differences among LPSs from different bacterial serotypes and their mutants include variations mainly within the composition and length of their O-specific polysaccharide chains.Proper assignation of the S or R chemotypes of endotoxins is possible by analyzing their electrophoretic profiles. The recent microchip electrophoretic methods provide fast characterizations and differentiations of endotoxins. The methods are applicable for determination directly from whole-cell lysates after destruction of the proteinaceous components by proteinase K digestion and precipitation of the LPS components. The partially purified LPS components are visualized either by interaction with dodecyl sulfate and a fluorescent dye, or by a covalently bound fluorescent dye. These chip electrophoretic methods have advantages of high speed and quantification and replace the sodium dodecyl sulfate-polyacrylamide gel electrophoresis with silver staining.
Asunto(s)
Electroforesis por Microchip/métodos , Lipopolisacáridos/análisis , Lipopolisacáridos/química , Electroforesis en Gel de Poliacrilamida/métodos , Endopeptidasa K/análisis , Colorantes Fluorescentes/metabolismo , Bacterias Gramnegativas/clasificación , Bacterias Gramnegativas/aislamiento & purificación , Antígenos O/análisis , Antígenos O/química , Serotipificación/métodos , Tinción con Nitrato de Plata/métodos , Dodecil Sulfato de Sodio/metabolismoRESUMEN
The structural variations in the rough-type endotoxins [lipopolysaccharides (LPSs)] of Shigella sonnei mutant strains (S. sonnei phase II-4303, R41, 562H and 4350) were investigated by Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and tandem MS. A series of S. sonnei mutants had previously been the subject of analytical studies on the biosynthesis of heptose components in the core oligosaccharide region of LPSs. This study gives a complete overview on the structures of the full core and lipid A of S. sonnei mutant strains by MS. We found that the LPSs of the isogenic rough mutants were formed in a step-like manner containing 0:1:2:3 heptose in the deep core region of 4350, 562H, R41 and 4303, respectively, and the longest LPS from the mutant S. sonnei 4303 contained also five hexoses. The structural variations in the lipid A moiety and in the oligosaccharide part of the intact LPS were followed by MALDI-TOF-MS/MS. For the dissolution and the ionization of the samples, 2,5-dihydroxybenzoic acid in citric acid solution was applied as matrix. The detailed evaluation of the mass spectra indicates heterogeneity in the lipid part due to the differences in the phosphate and fatty acid composition.
Asunto(s)
Endotoxinas/química , Shigella sonnei/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Conformación MolecularRESUMEN
A fast microchip electrophoresis method was developed to analyze and differentiate bacterial endotoxins directly from whole-cell lysates after removal of the proteinaceous components with proteinase K digestion and a precipitation of the endotoxin components. The partially purified endotoxin components were visualized by the interaction with dodecyl sulphate and then a fluorescent dye. The lipopolysaccharide (LPS) profiles can be directly evaluated from digested bacterial cells, and the electrophoresis patterns very closely resembled to those of pure LPSs, and the R and S chemotypes can be used to assign the strains. The method has been found to be useful in the screening of a large number of bacterial mutants and the structural characterization of endotoxins extracted only from 1 ml cultures.
Asunto(s)
Electroforesis por Microchip/métodos , Enterobacteriaceae/química , Lipopolisacáridos/análisis , Serotipificación/métodos , Enterobacteriaceae/clasificación , Lipopolisacáridos/clasificaciónRESUMEN
A novel microchip electrophoresis method was developed and applied for sensitive detection and quantitative analysis of endotoxins extracted from Gram-negative bacteria. The method provides a fast and quantitative differentiation of smooth and rough endotoxins based on the solubilization and complexation of the lipopolysaccharides with dodecylsulfate, and then with a fluorescent dye. The migration of the complexes was followed by LIF detection. The novel method is able to replace the SDS-PAGE with the advantage of high speed and better sensitivity, and by avoiding the laborious gel-preparation and silver staining.
Asunto(s)
Electroforesis por Microchip/métodos , Endotoxinas/análisis , Electroforesis en Gel de PoliacrilamidaRESUMEN
A new CE method for fast and efficient analysis of bacterial endotoxins (lipopolysaccharides) is described. It is based on the strong interaction between proteins and endotoxins. The UV absorption of the protein component in the complex is used for the detection. The electrophoretic mobility of the complex hemoglobin/endotoxin can be employed for qualitative analysis of the endotoxin. For instance, the structural differences between "smooth" and "rough" lipopolysaccharides from Salmonella minnesota (wild-type), Salmonella minnesota R595 and Shigella sonnei R562H are reflected in the electrophoretic mobilities of their hemoglobin complex.